The regulation of cell volume is of critical importance to the CNS due to the restrictions of the skull. Brain swelling, which may occur in response to a lowering of plasma osmolarity or during cytotoxic edema, is associated with a number of clinical conditions, including congestive heart failure, hepatic encephalopathy, ischemic stroke, or head trauma. To counteract the increased volume, cells release Kv, CI-, and "non-perturbing" organic osmolytes, a major component of which is myo-inositol. Efflux of the osmolytes occurs via a volume-sensitive organic anion channel (VSOAC), which primarily gates CI'. Although most attention has been focused on the role played by glia in the process of volume regulation, cultured neuronal cells have also been recently shown to exhibit similar properties. Moreover, recent results from this laboratory indicate that high concentrations of myo-inositol are present in some neuronal populations and that the polyol can be released in a volume-dependent manner. Although the electrophysiological and pharmacological characteristics of VSOAC have been well documented, relatively little is known of the cell signaling pathways that regulate osmolyte efflux through this channel. A central tenet of this proposal is that, in the face of hypoosmotic challenge, the capacity of neural cells to restore their volume via the efflux of inositol and other osmolytes can be regulated by extracellular agonists operating via phosphoinositide-linked receptors, such as the muscarinic cholinergic receptor. Thus we plan to examine the characteristics of myo-inositol efflux from human SH-SY5Y neuroblastoma cells under hypoosmotic conditions and evaluate the relationship between effiux of the polyol and changes in cell volume. In addition, we will test the hypothesis that activation of muscarinic cholinergic and other phosphoinositide-linked receptors leads to an increase in the effiux of inositol, and other osmolytes, from these cells. Furthermore, the possibility that individual osmolytes exit the cell via multiple VSOACs will be explored via a comparison of the effiux characteristics of inositol, taurine, and D-aspartate, all of which can be released from these cells. The ability to manipulate osmolyte effiux could be of potential benefit for a number of clinically relevant conditions. Accordingly, knowledge of the signal transduction pathways that regulate VSOAC is an essential prerequisite for the rational design of therapeutic agents.